In recent years, a number of methods have been developed for manipulating DNA. Some of these methods employ biomolecules to cut or cleave DNA, which in some instances renders the substrate DNA nonfunctional. Other methods employ biomolecules to facilitate insertion of new pieces of nucleic acid into the cleavage site of the DNA substrate. The insertion of new segments of nucleic acid into the cleavage sites of the DNA substrate changes the characteristics of the RNA or protein molecules encoded by the substrate DNA molecules. Accordingly, the biomolecules which catalyze the cleavage of DNA substrates or the insertion of new nucleic acid molecules into the DNA substrates are useful tools for genetic engineering, for analytical studies and for diagnostic studies. One such molecule used for cleaving DNA substrates is the restriction endonuclease.
Restriction endonucleases are enzymatic proteins that cleave double-stranded DNA. Such endonucleases recognize specific nucleotide sequences in double-stranded DNA, and cleave both strands within or near the specific recognition site. Such specificity renders the restriction endonucleases important tools in the controlled fragmentation of double-stranded DNA. Restriction endonucleases are also useful analytical tools for determining whether certain sequences are present in substrate DNA and in genomic sequencing studies.
However, restriction endonucleases only cleave DNA substrates; they do not insert new nucleic acid molecules into the cleaved DNA substrate. Accordingly, another biomolecule is needed to insert new pieces of DNA or RNA into the double-stranded DNA.
Ribozymes are catalytic RNA molecules that cleave RNA and, in certain circumstances, that insert new pieces of RNA into the cleavage site of the RNA substrate. Unfortunately, ribozymes have not been particularly useful for cleaving single-stranded DNA substrates or double-stranded DNA substrates. Ribozymes cut single-stranded DNA only under extreme conditions of elevated temperatures and high concentrations of magnesium. Ribozymes can be used to cleave double-stranded DNA only after the DNA is denatured and separated into two pieces of single-stranded DNA. Moreover, ribozymes have limited use in systems containing ribonucleases.
Accordingly, it is desirable to have new methods that employ a new tool that is capable of cleaving double-stranded DNA molecules, single-stranded DNA molecules, and single-stranded RNA molecules at specific sites. Methods which employ a new biomolecule capable of cleaving RNA molecules, single-stranded DNA molecules and double-stranded DNA molecules at specific sites and simultaneously inserting a new nucleic acid molecule into the cleavage site are especially desirable.